Targeted/immunomodulatory fusion proteins and methods for making same

ABSTRACT

The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences encoding the fusion proteins, wherein the chimeric fusion proteins comprises at least one targeting moiety and at least one immunomodulatory moiety that counteracts the immune tolerance of cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of and claims priority toco-pending U.S. patent application Ser. No. 15/795,404 filed on Oct. 27,2017, now U.S. Pat. No. ______, which in turn claimed priority toco-pending U.S. patent application Ser. No. 15/047,062 filed on Feb. 18,2016, now U.S. Pat. No. 9,809,651 issued on Nov. 7, 2017, which in turnclaimed priority to copending U.S. patent application Ser. No.14/458,674 filed on Aug. 13, 2014, now U.S. Pat. No. 9,340,617 issuedMay 17, 2016, which in turn claimed priority to copending U.S. patentapplication Ser. No. 13,799,409 filed on Mar. 13, 2013, now U.S. Pat.No. 8,815,247 issued on Aug. 26, 2014, which in turn claimed priority toco-pending Indian Patent Application No. 1689/CHE/2012 filed on Apr. 30,2012 and Indian Patent Application No. 1690/CHE/2012 filed on Apr. 30,2012, the contents of all are hereby incorporated by reference hereinfor all purposes.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates generally to the field of generatingfusion proteins to be used in cancer therapy, and more specifically, tonucleotide sequences encoding the fusion proteins, wherein the fusion orchimeric polypeptides comprises at least one targeting moiety and atleast one immunomodulatory moiety that counteracts the immune toleranceof cancer cells.

Related Art

The immune system provides the human body with a means to recognize anddefend itself against microorganisms and substances recognized asforeign or potentially harmful. While passive immunotherapy of cancerwith monoclonal antibodies and passive transfer of T cells to attacktumor cells have demonstrated clinical efficacy, the goal of activetherapeutic vaccination to induce these immune effectors and establishimmunological memory against tumor cells has remained challenging.Several tumor-specific and tumor-associated antigens have beenidentified, yet these antigens are generally weakly immunogenic andtumors employ diverse mechanisms to create a tolerogenic environmentthat allows them to evade immunologic attack. Strategies to overcomesuch immune tolerance and activating robust levels of antibody and/or Tcell responses hold the key to effective cancer immunotherapy. Moreimportant, the individual proteins and how to create an active chimericpolypeptide with an active tertiary structure needs to be explored.

SUMMARY OF THE INVENTION

The present invention provides polynucleotides, as well as polypeptidesencoded thereby, that are expressed in cancer cells. Thesepolynucleotides and expressed polypeptides are useful in a variety oftherapeutic methods for the treatment of cancer. The present inventionfurther provides methods of reducing growth of cancer cells bycounteracting immune tolerance of cancer cells, wherein T cell remainactive and inhibit the recruitment of T-regulatory that are known tosuppress the immune system's response to the tumor. Thus the chimericpolypeptides generated by the polynucleotides sequences of the presentinvention are useful for treating cancer because of the expressed fusionor chimeric polypeptides.

In one aspect, the present invention provides for chimeric polypeptidescontaining at least one targeting moiety to target a cancer cell and atleast one immunomodulating moiety that counteracts immune tolerance ofcancer cell, wherein the targeting moiety and the immunomodulatingmoiety are linked by a amino acid spacer of sufficient length of aminoacid residues so that both moieties can successfully bond to theirindividual target. In the alternative, the targeting moiety and theimmunomodulating moiety that counteract immune tolerance of cancer cellmay be bound directly to each other. The chimeric/fusion polypeptides ofthe invention are useful for binding to a cancer cell receptor andreducing the ability of cancer cells to avoid an immune response.

The present invention is based on preparing chimeric/fusion proteins byexpression of polynucleotides encoding the fusion proteins thatcounteract or reverse immune tolerance of cancer cells. Cancer cells areable to escape elimination by chemotherapeutic agents or tumor-targetedantibodies via specific immunosuppressive mechanisms in the tumormicroenvironment and such ability of cancer cells is recognized asimmune tolerance. Such immunosuppressive mechanisms includeimmunosuppressive cytokines (for example, Transforming growth factorbeta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloiddendritic cells (DCs). By counteracting tumor-induced immune tolerance,the present invention provides effective compositions and methods forcancer treatment, optional in combination with another existing cancertreatment. The present invention provides strategies to counteracttumor-induced immune tolerance and enhance the antitumor efficacy ofchemotherapy by activating and leveraging T cell-mediated adaptiveantitumor against resistant or disseminated cancer cells.

In another aspect, the present invention provides a molecule includingat least one targeting moiety fused with at least one immunomodulatorymoiety. The targeting moiety specifically binds a target molecule, andthe immunomodulatory moiety specifically binds one of the followingmolecules: (i) Transforming growth factor-beta (TGF-β): (ii) Programmeddeath-1 ligand 1 (PD-L1) or Programmed death-1 ligand 2 (PD-L2); (iii)Receptor activator of nuclear factor-KB (RANK) ligand (RANKL); (iv)Transforming growth factor-beta receptor (TGF-pR); (v) Programmeddeath-1 (PD-1); (vi) 4-1BB receptor or (vii) Receptor activator ofnuclear factor-κB (RANK).

In a further aspect, the targeting moiety includes an antibody, antibodyfragment including the light or heavy chains of the antibody, scFv, orFc-containing polypeptide that specifically binds a component of a tumorcell, tumor antigen, tumor vasculature, tumor microenvironment, ortumor-infiltrating immune cell. Preferably, the targeting moiety is anantibody or a fragment thereof having binding affinity for a componenton a tumor cell. Notably each of the heavy chain and light chain mayindividually be linked to a separate and distinct immunomodulatorymoiety. Further, a heavy or light chain of an antibody targeting moietymay be linked to an immunomodulatory moiety which in turn can be furtherlinked to a second immunomodulatory moiety wherein there is a linkerbetween the two immunomodulatory moieties.

In a still further aspect, there is provided a chimeric polypeptide thatcomprised a tumor targeting moiety and an immunomodulatory moietycomprising a molecule that binds transforming growth factor beta(TGF-β), wherein the tumor targeting moiety is an antibody that binds toEGFR1, where in the antibody can be the full antibody, heavy chain orlight chain. The tumor targeting moiety may include monoclonalantibodies that target a cancer cell, including but not limited tocetuximab, trastuzumab, ritubximab, ipilimumab, tremelimumab,muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab,infliximab. gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan,adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab,bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101(Aphton), volociximab (Biogen Idec and PDL BioPharm), Anti-CD80 mAb(Biogen Idec), Anti-CD23 mAb (Biogen Idel), CAT-3888 (Cambridge AntibodyTechnology), CDP-791 (Imclone), eraptuzumab (Immunomedics), MDX-010(Medarex and BMS), MDX-060 (Medarex), MDX-070 (Medarex), matuzumab(Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics),zanolimumab (Serono and Genmab), adecatumumab (Sereno), oregovomab(United Therapeutics), nimotuzumab (YM Bioscience), ABT-874 (AbbottLaboratories), denosumab (Amgen), AM 108 (Amgen), AMG 714 (Amgen),fontolizumab (Biogen Idec and PDL BioPharm), daclizumab (Biogent Idecand PDL BioPharm), golimumab (Centocor and Schering-Plough), CNTO 1275(Centocor), ocrelizumab (Genetech and Roche), HuMax-CD20 (Genmab),belimumab (HGS and GSK), epratuzumab (Immunomedics), MLN1202 (MillenniumPharmaceuticals), visilizumab (PDL BioPharm), tocilizumab (Roche),ocrerlizumab (Roche), certolizumab pegol (UCB, formerly Celltech),eculizumab (Alexion Pharmaceuticals), pexelizumab (AlexionPharmaceuticals and Procter & Gamble), abciximab (Centocor),ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox), or MYO-029(Wyeth).

In another aspect, the tumor targeting moiety is a monoclonal antibodythat binds to HER2/Neu, CD20, CTLA4, EGFR1 and wherein the antibody canbe the full antibody, heavy chain or light chain.

In yet another aspect, the targeting moiety is a molecule thatspecifically binds epidermal growth factor receptor (EGFR1, Erb-B 1),HER2/neu (Erb-B2), CD20, cytotoxic T-lymphocyte antigen-4 (CTLA-4) whichis essential for Treg function (CD 152); H-land Interleukin-6 (IL-6).

In a still further aspect, the targeting moiety specifically binds acomponent of a regulatory T cell (treg), myeloid suppressor cell, ordendritic cell. In another aspect, the targeting moiety specificallybinds one of the following molecules: (i) CD4; (ii) CD25 (IL-2ctreceptor; IL-2aR); (iii) Transforming growth factor-beta receptor(TGF-pR); (vi) Transforming growth factor-beta (TGF-β): (vii) ProgrammedDeath-1 (PD-1); (viii) Programmed death-1 ligand (PD-LI or PD-L2.

In another aspect, the immunomodulatory moiety specifically binds one ofthe following molecules: (i) Transforming growth factor-beta (TGF-β):(ii) Programmed death-1 ligand (PD-L1 or PD-L2); or 4-1BB receptor.

In yet another aspect, the immunomodulatory moiety includes a moleculethat binds TGF-β and inhibits the function thereof. Specifically theimmunomodulatory moiety includes an extracellular ligand-binding domainof Transforming growth factor-beta receptor TGF-βRII, TGF-βRIIb, orTGF-βRIII. In another aspect the immunomodulatory moiety includes anextracellular ligand-binding domain (ECD) of TGF-βRII. Still further theimmunomodulatory moiety may include H-4-1BB ligand which binds to the4-1BB receptor to stimulate T-cells to help eradiate tumor.

In a still further aspect, the targeting moiety includes an antibody,antibody fragment, or polypeptide that specifically binds to HER2/neu,EGFR1, CD20, or cytotoxic T-lymphocyte antigen-4 (CTLA-4) and whereinthe immunomodulatory moiety includes an extracellular ligand-bindingdomain of TGF-βRII.

In yet another aspect, the immunomodulatory moiety includes a moleculethat specifically binds to and inhibit the activity of Programmeddeath-1 ligand 1 (PD-L 1) or Programmed death-1 ligand 2 (PD-L2). Inanother aspect, the immunomodulatory moiety includes an extracellularligand-binding domain or ectodomain of Programmed Death-1 (PD-1).

In a further aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD25 (IL-2a receptor;IL-2aR), or CD4 and wherein, the immunomodulatory moiety includes anextracellular ligand-binding domain or ectodomain of Programmed Death-1(PD-1).

In a still further aspect, the targeting moiety includes an antibody orantibody fragment that specifically binds to CD20, and theimmunomodulatory moiety includes a sequence from transforming growthfactor-β (TGF-β).

In one aspect, the present invention provides for optimized genesencoding for a fusion polypeptide comprising at least one targetingmoiety and at least one immunomodulatory moiety for treating cancer in ahuman subject wherein the optimized genes have been modified to increaseexpression in a human subject. preferably the optimized genes comprisesequences for encoding a targeting moiety or an immunomodulatory moietyselected from SEQ ID NOs: 12 to 28.

In another aspect, the present invention provides for a vectorcomprising optimized genes for treating cancer in a human subjectwherein the optimized genes have been modified to increase CG sequences.Preferably, the vector includes sequences for encoding at least onetargeting moiety and at least one immunomodulatory moiety selected fromSEQ ID NOs: 12 to 28.

In yet another aspect, the present invention provides for a method oftreating cancer in a subject, the method comprising:

-   -   a. providing at least one recombinant vector comprising        nucleotide sequences that encode at least one targeting moiety        and at least one immunomodulatory moiety selected from SEQ ID        NOs: 12 to 28; and    -   b. administering the recombinant vector to the subject under        conditions such that said nucleotide sequences are expressed at        a level which produces a therapeutically effective amount of the        encoded fusion proteins in the subject.

In an alternative aspect, the present invention provides an expressionvector comprising polynucleotides of optimized genes that encode atleast one targeting moiety and at least one immunomodulatory moietyselected from SEQ ID NOs: 12 to 28.

In yet another aspect, the present invention provides a recombinant hostcell transfected with a polynucleotide that encodes a fusion proteinpeptide of the present invention.

In a still further aspect, the present invention contemplates a processof preparing a fusion protein of the present invention comprising:

-   -   a. transfecting a host cell with polynucleotide sequences that        encode chimeric fusion proteins to produce a transformed host        cell, wherein the polynucleotide sequences encode at least one        targeting moiety and at least one immunomodulatory moiety        selected from SEQ ID NOs: 12 to 28; and    -   b. maintaining the transformed host cell under biological        conditions sufficient for expression of the peptide.

In another aspect, the present invention relates to the use of achimeric fusion protein, as shown in FIGS. 1 to 15, in the use of amedicament for the treatment of cancer. Preferably, the fusion proteinis expressed in a host cell and such expressed proteins are administeredin a therapeutic amount to reduce the effects of cancer in a subject inneed thereof.

In a still further aspect, the present invention provides a method ofpreventing or treating a neoplastic disease. The method includesadministration to a subject in need thereof one or more fusion proteinsof the invention, in various aspects, the subject is administered one ormore molecule of the invention in combination with another anticancertherapy, in one aspect, the anticancer therapy includes achemotherapeutic molecule, antibody, small molecule kinase inhibitor,hormonal agent or cytotoxic agent. The anticancer therapy may alsoinclude ionizing radiation, ultraviolet radiation, cryoablation, thermalablation, or radiofrequency ablation.

In yet another aspect, the present invention provides for a method ofpreparing therapeutically active antibody-peptide fusion proteins, themethod comprising;

-   -   a. preparing a codon optimized sequence of the said fusion        protein;    -   b. cloning the optimized sequence of said fusion protein in a        host cell capable of transient or continued expression;    -   c. growing the host cell in a media under suitable conditions        for growing and allowing the host cell to express the cloned        protein; and    -   d. subjecting the expressed protein to purification and        optionally checking the bi-specific binding capabilities of the        protein to its targets.

In a preferred embodiment the therapeutically active antibody-peptidefusion proteins is a targeting antibody fused to one or moreimmunomodulating moiety that counteracts immune tolerance of a cancercell. In one aspect, the immunomodulating moiety may be linked by anamino acid spacer of sufficient length to allow bi-specific binding ofthe molecule. The immunomodulating moiety may be bound to either theC-terminus of the heavy or light chain of the antibody

In a preferred method as described above, the immunomodulating moiety is(i) Transforming growth factor-beta (TGF-β), (ii) Programmed death-1(PD-1), (iii) CTLA-4 or (iv) 4-1BB or parts thereof and the targetingantibody binds epidermal growth factor receptor (EGFR1, Erb-B 1),HER2/neu (Erb-B2), CD20, CD6, CTLA-4, Mucin 1 (MUC-1), Interleukin-2(IL-2) or Interleukin-6 (IL-6).

The method of the present invention provides nucleotide sequences thatencode the therapeutically active antibody-peptide fusion proteins andsuch expression may be conducted in a transient cell line or a stablecell line. The transient expression is accomplished by transfecting ortransforming the host with vectors carrying the fusion proteins intomammalian host cells

Once the fusion peptides are expressed, they are preferably subjected topurification and in-vitro tests to check its bi-specificity, that being,having the ability to bind to both the target moiety andimmunomodulating moiety. Such tests may include in-vitro test such asELISA or NK/T-cell binding assays to validate bi-functional targetbinding or immune cell stimulation.

Notably once the specific fusion peptides demonstrate the desiredbi-specificity, such fusion peptides are selected for sub-cloning into astable cell line for larger scale expression and purification. Suchstable cell lines are previously disclosed, such as a mammalian cellline, including but not limited to HEK293, CHO or NSO.

In a further aspect, the culture medium can be improved by additions tosuch medium. For example, the culture medium may include a divalenttransitional metallic salt which is added to the cell culture eitherinitially or in fed-batch mode to reduce accumulation of lactate duringculturing and/or reduce heterogeneity of the fusion proteins. Adesirable transitional metallic salt includes a zinc ion and theaddition of the metal ion may be carried out during different phases ofthe production.

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of with the amino acid sequence ofAnti-HER2/neu-TGFβRII fusion protein at LC constant region with theamino acid sequence of anti-HER2/neu heavy chain (SEQ ID NO: 1) andanti-HER2/neu light chain (SEQ ID NO: 2) attached to amino residues forTGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters and wherein a linker (SEQ ID NO: 3) is positioned between theanti-HER2/neu light chain and TGF-βRII and shown in italics.

FIG. 2 shows the amino acid sequences of Anti-EGFR1-TGFβRII fusionprotein at LC constant region with amino acid sequence of Anti-EGFR1heavy chain (SEQ ID NO: 5) and the amino acid sequence of Anti-EGFR1light chain (SEQ ID NO: 6) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the Anti-EGFR1light chain and TGF-βRII and shown in italics.

FIG. 3 shows the amino acid sequences of Anti-CTLA4-TGFβRII fusionprotein at LC constant region with amino acid sequence of anti-CTLA4heavy chain (SEQ ID NO: 7) and amino acid sequence of anti-CTLA4 lightchain (SEQ ID NO: 8) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the anti-CTLA4light chain and TGF-βRII and shown in italics.

FIG. 4 shows the amino acid sequences of Anti-HER2/neu HC-4-1BB andLC-TGFβRII fusion protein with amino acid sequence ofAnti-HER2/neu/HC-4-1BB fusion protein wherein the amino acid sequencefor Anti-HER2/neu heavy chain (SEQ ID NO: 1) is attached to a linker(SEQ ID NO: 3) shown in italics and the sequence for 4-1BB(immunomodulatory moiety) (SEQ ID NO: 9) is in written text font andamino acid sequence of anti-HER2/neu light chain (SEQ ID NO: 2) attachedto amino residues for TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4)identified in bold letters and wherein a linker (SEQ ID NO: 3) ispositioned between the anti-HER2/neu light chain and TGF-βRII and shownin italics.

FIG. 5 shows the amino acid sequence of Anti-EGFR1 HC-4-1BB andLC-TGFβRII fusion protein with amino acid sequence of Anti-EGFR1 heavychain-4-1BB fusion protein wherein the amino acid sequence forAnti-EGFR1 heavy chain (SEQ ID NO: 5) is attached to a linker (SEQ IDNO: 3) is shown in italics and the sequence for 4-1BB (immunomodulatorymoiety) (SEQ ID NO: 9) is in written text font and amino acid sequenceof light chain Anti-EGFR1 (SEQ ID NO: 6) attached to amino residues forTGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 6 shows the amino acid sequence of Anti-CTLA4 HC-4-1BB andLC-TGFβRII fusion protein with amino acid sequence of Anti-CTLA4 heavychain-4-1BB fusion protein wherein the amino acid sequence forAnti-CTLA4 heavy chain (SEQ ID NO: 7) is attached to a linker (SEQ IDNO: 3) is shown in italics and the sequence for 4-1BB (immunomodulatorymoiety) (SEQ ID NO: 9) is in written text font and amino acid sequenceof Anti-CTLA4 light chain (SEQ ID NO: 8) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 7 shows the amino acid sequence of Anti-HER2/neu HC-PD1 andLC-TGFβRII fusion protein with amino acid sequence of Anti-HER2/neuheavy chain-PD1 fusion protein wherein the amino acid sequence for theAnti-HER2/neu heavy chain (SEQ ID NO: 1) is attached to a linker (SEQ IDNO: 3) is shown in italics and the sequence for PD1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font and amino acid sequenceof Anti-HER2/neu light chain (SEQ ID NO: 2) is attached to aminoresidues for TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4)identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

FIG. 8 shows the amino acid sequence of Anti-EGFR1 HC-PD1 and LC-TGFβRIIfusion protein with amino acid sequence of Anti-EGFR1 heavy chain-PD1fusion protein wherein the amino acid sequence Anti-EGFR1 heavy chain(SEQ ID NO: 5) is attached to a linker (SEQ ID NO: 3) shown in italicsand the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is inwritten text font and amino acid sequence of Anti-EGFR1 light chain (SEQID NO: 6) attached to amino residues for TGF-βRII (immunomodulatorymoiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ IDNO: 3) therebetween.

FIG. 9 shows the amino acid sequence of Anti-CTLA4 HC-PD1 and LC-TGFβRIIfusion protein with amino acid sequence of Anti-CTLA4 heavy chain-PD1fusion protein wherein the amino acid sequence Anti-CTLA4 heavy chain(SEQ ID NO: 7) is attached to a linker (SEQ ID NO: 3) shown in italicsand the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is inwritten text font and amino acid sequence of Anti-CTLA4 light chain (SEQID NO: 8) attached to amino residues for TGF-βRII (immunomodulatorymoiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ IDNO: 3) therebetween.

FIG. 10 shows the amino acid sequence of Anti-HER2/neu HC-TGFβRII-4-1BBfusion protein with amino acid sequence of Anti-HER2/neu heavychain-TGFβRII-4-1BB fusion protein wherein the amino acid sequence forAnti-HER2/neu heavy chain (SEQ ID NO: 37) is attached to a linker (SEQID NO: 3) shown in italics and the sequence for TGFβRII(immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold lettersand the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ IDNO: 9) is in written text font with linker between (SEQ ID No: 11) andincluding the amino acid sequence of Anti-HER2/neu light chain (SEQ IDNO: 2).

FIG. 11 shows the amino acid sequence of Anti-EGFR1 HC-TGFβRII-4-1BBfusion protein with amino acid sequence of Anti-EGFR1 heavychain-TGFβRII-4-1BB fusion protein wherein the amino acid sequence forAnti-EGFR1 heavy chain (SEQ ID NO: 38) sequence is attached to a linker(SEQ ID NO: 3) shown in italics and the sequence for TGFβRII(immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold lettersand the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ IDNO: 9) is in written text font with linker between (SEQ ID NO: 11) andincluding the amino acid sequence of Anti-EGFR1 light chain (SEQ ID NO:6).

FIG. 12 shows the amino acid sequence of Anti-CTLA4 HC-TGFβRII-4-1BBfusion protein with amino acid sequence of Anti-CTLA4 heavychain-TGFβRII-4-1BB fusion protein wherein the amino acid sequenceAnti-CTLA4 heavy chain (SEQ ID NO: 39) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for TGFβRII (immunomodulatorymoiety) (SEQ ID NO: 4) is identified in bold letters and the amino acidsequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is inwritten text font with linker between (SEQ ID NO: 11) and including theamino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8).

FIG. 13 shows the amino acid sequence of Anti-HER2/neu HC-TGFβRII-PD1fusion protein with amino acid sequence of Anti-HER2/neu heavychain-TGFβRII-PD1 fusion protein wherein the amino acid sequenceAnti-HER2/neu heavy chain (SEQ ID NO: 37) is attached to a linker (SEQID NO: 3) shown in italics and the sequence for TGFβRII(immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold lettersand the amino acid sequence for PD-1 (immunomodulatory moiety) (SEQ IDNO: 10) is in written text font with linker between (SEQ ID No: 11) andincluding the amino acid sequence of Anti-HER2/neu light chain (SEQ IDNO: 2).

FIG. 14 shows the amino acid sequence of Anti-EGFR1 HC-TGFβRII-PD1fusion protein with amino acid sequence of Anti-EGFR1 heavychain-TGFβRII-PD1 fusion protein wherein the amino acid sequenceAnti-EGFR1 heavy chain (SEQ ID NO: 38) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for TGFβRII (immunomodulatorymoiety) (SEQ ID NO: 4) is identified in bold letters and the amino acidsequence for PD-1 (immunomodulatory moiety) (SEQ ID NO: 10) is inwritten text font with linker between (SEQ ID No: 11) and including theamino acid sequence of Anti-EGFR1 light chain (SEQ ID NO: 6).

FIG. 15 shows the of Anti-CTLA4 HC-TGFβRII-PD1 fusion protein with aminoacid sequence of Anti-CTLA4 heavy chain-TGFβRII-PD1 fusion proteinwherein the amino acid sequence Anti-CTLA4 heavy chain (SEQ ID NO: 39)is attached to a linker (SEQ ID NO: 3) shown in italics and the sequencefor TGFβRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified inbold letters and the amino acid sequence for PD-1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font with linker between (SEQID NO: 11) and including the amino acid sequence of Anti-CTLA4 lightchain (SEQ ID NO: 8).

FIG. 16 shows the nucleotide sequence of Anti-HER2/neu heavy chainconstant region with linker (SEQ ID NO: 12) and TGFβRII ECD (SEQ ID NO:13) that have been codon optimized for expression in CHO cell.

FIG. 17 shows the nucleotide sequence of Anti-HER2/neu heavy chainvariable region (SEQ ID NO: 14), Anti-HER2/neu light chain variableregion (SEQ ID NO: 15) and Anti-EGFR1 heavy chain constant region withlinker (SEQ ID NO: 16) that have been codon optimized for expression inCHO cell.

FIG. 18 shows the nucleotide sequence of Anti-EGFR1 heavy chain variableregion (SEQ ID NO: 17), Anti-EGFR1 light chain variable region (SEQ IDNO: 18), Anti-CTLA4 heavy chain variable region (SEQ ID NO: 19) andAnti-CTLA4 light chain variable region (SEQ ID NO: 20) that have beencodon optimized for expression in CHO cell.

FIG. 19 shows the nucleotide sequence of Anti CD20 IgG1 molecule (SEQ IDNO: 21), Anti-CD20 heavy chain variable region (SEQ ID NO: 22) andAnti-CD20 light chain variable region (SEQ ID NO: 23) that have beencodon optimized for expression in CHO cell.

FIG. 20 shows the nucleotide sequence of 4-1BB (SEQ ID NO: 24) andAnti-IL6R heavy chain (SEQ ID NO: 25) that have been codon optimized forexpression in CHO cell.

FIG. 21 shows the nucleotide sequence of Anti-IL6R light chain variableregion (SEQ ID NO: 26), Anti-4-1BB heavy chain (SEQ ID NO: 27) andAnti-4-1BB light chain variable region (SEQ ID NO: 28) that have beencodon optimized for expression in CHO cell.

FIG. 22 shows the analysis of Protein A purified Anti-HER2/neu-TGFβRIIand Anti-EGFR1-TGFβRII at 12% PAGE

FIG. 23 A shows Anti-HER2/neu-TGFβRII samples analyzed by Protein A/SECChromatography and B Anti-EGFR1-TGFβRII samples analyzed by ProteinA/SEC Chromatography.

FIG. 24 A shows that Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRIImolecules bind to the TGFβ indicating that the fusion protein isfunctional and B shows that Anti-HER2-TGFβRII inhibits the proliferationof BT474 cell line similar to the Bmab200 (Herceptin).

FIG. 25 shows that Anti-EGFR1-TGFβRII-inhibits the proliferation of A431cell line similar to the Cetuximab.

FIG. 26 shows the ADCC activity of Anti-HER2-TGFβRII on BT474 cells issimilar to that of Bmab200 (Herceptin).

FIG. 27 shows the ADCC activity of Anti-EGFR1-TGFβRII on A431 cellswherein the ADCC activities are similar to that of Cetuximab.

FIG. 28 shows the ADCC activity of ADCC activity of Anti-EGFR1-4-1BB incomparison with Anti-EGFR1-TGFβRII and cetuximab.

FIG. 29 A shows that the binding activity of Anti-CTLA4-TGFβRII to TGFβ1is comparable to Anti-EGFR1-TGFβRII and B shows that the bindingactivity of Anti-CTLA4-TGFβRII to CTLA4.

FIG. 30 A shows the binding activity of Anti-CTLA4-TGFβRII to determinethe level of PD1-Fc binding and B shows the binding activity ofAnti-EGRF1-4-1BB to determine the binding of 4-1BBL.

FIG. 31 A shows the binding activity of Anti-EGFR1-4-1BB to EGFR and Bshows the binding activity of PD1-Fc-4-1BB to find out PDL1-Fc.

FIG. 32 shows the binding activity of Anti-EGFR1-PD1 to EGFR and PD1.

FIG. 33 shows photographs of expressed proteins and reduction alkylationthereof.

FIG. 34 A shows the mass spectrum Mass Spectrum of light chain (LC)(Reduced) of Anti-HER2/neu-TGFβRII ECD fusion and B shows DeconvolutedMass Spectrum of LC (Reduced) of Anti-HER2/neu-TGFβRII ECD fusion.

FIG. 35 shows the Mass Spectrum of heavy chain (HC) (Reduced) ofAnti-HER2/neu-TGFβRII ECD fusion.

FIG. 36 A shows the Mass Spectrum of LC (Reduced) of Anti-EGFR1-TGFβRIIECD and B shows the Deconvoluted Mass Spectrum of LC (Reduced) ofAnti-EGFR1-TGFβRII ECD.

FIG. 37 shows the Mass Spectrum of HC (Reduced) of Anti-EGFR1-TGFβRIIECD.

FIG. 38 A shows the UV Chromatogram of Tryptic Peptides ofAnti-HER2/neu-TGFβRII ECD fusion protein and B shows the Total IonChromatogram (TIC) of Tryptic Peptides of Anti-HER2/neu-TGFβRII ECDfusion protein.

FIGS. 39, 40 and 41 provide lists of expected/observed tryptic peptideof the light chain, heavy chain and linked motif of theAnti-HER2/neu-TGFβRII ECD fusion protein, respectively.

FIG. 42 A shows the UV Chromatogram of Tryptic Peptides ofAnti-EGFR1-TGFβRII ECD fusion protein and B shows the Total IonChromatogram (TIC) of Tryptic Peptides of Anti-EGFR1-TGFβRII ECD fusionprotein.

FIG. 43 provides a list of expected/observed tryptic peptide of thelight chain of the Anti-EGFR1-TGFβRII ECD fusion protein.

FIG. 44 shows the list of expected/observed tryptic peptide of the heavychain of the Anti-EGFR1-TGFβRII ECD fusion protein.

FIG. 45 shows the list of expected/observed tryptic peptide of the heavychain of the Anti-EGFR1-TGFβRII ECD fusion protein.

FIG. 46 shows the amino acid sequences of Cantuzumab—TGFβRII fusionprotein at LC constant region with amino acid sequence of Cantuzumabheavy chain (SEQ ID NO: 29) and amino acid sequence of Cantuzumab lightchain (SEQ ID NO: 30) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the Cantuzumablight chain and TGF-βRII and shown in italics.

FIG. 47 shows the amino acid sequences of Cixutumumab-TGFβRII fusionprotein at LC constant region with amino acid sequence of Cixutumumabheavy chain (SEQ ID NO: 31) and amino acid sequence of Cixutumumab lightchain (SEQ ID NO: 32) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the Cixutumumablight chain and TGF-βRII and shown in italics.

FIG. 48 shows the amino acid sequences of Clivatuzumab-TGFβRII fusionprotein at LC constant region with amino acid sequence of Clivatuzumabheavy chain (SEQ ID NO: 33) and amino acid sequence of Clivatuzumablight chain (SEQ ID NO: 34) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the Clivatuzumablight chain and TGF-βRII and shown in italics.

FIG. 49 shows the amino acid sequences of Pritumumab-TGFβRII fusionprotein at LC constant region with amino acid sequence of Pritumumabheavy chain (SEQ ID NO: 35) and amino acid sequence of Pritumumab lightchain (SEQ ID NO: 36) attached to amino acid residues for TGF-βRII(immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters andwherein a linker (SEQ ID NO: 3) is positioned between the Pritumumablight chain and TGF-βRII and shown in italics.

FIG. 50 shows the amino acid sequence of Cantuzumab HC-4-1BB andLC-TGFβRII fusion protein wherein the amino acid sequence for theCantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ IDNO: 3) which is shown in italics and the sequence for 4-1BB(immunomodulatory moiety) (SEQ ID NO: 9) is in written text font andamino acid sequence of Cantuzumab light chain (SEQ ID NO: 30) isattached to amino residues for TGF-βRII (immunomodulatory moiety) (SEQID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3)therebetween.

FIG. 51 shows the amino acid sequence of Cixutumumab HC-4-1BB andLC-TGFβRII fusion protein wherein the amino acid sequence for theCixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for 4-1BB (immunomodulatorymoiety) (SEQ ID NO: 9) is in written text font and amino acid sequenceof Cixutumumab light chain (SEQ ID NO: 32) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 52 shows the amino acid sequence of Clivatuzumab HC-4-1BB andLC-TGFβRII fusion protein wherein the amino acid sequence for theClivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for 4-1BB (immunomodulatorymoiety) (SEQ ID NO: 9) is in written text font and amino acid sequenceof Clivatuzumab light chain (SEQ ID NO: 34) is attached to aminoresidues for TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4)identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

FIG. 53 shows the amino acid sequence of Pritumumab HC-4-1BB andLC-TGFβRII fusion protein wherein the amino acid sequence for thePritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for 4-1BB (immunomodulatorymoiety) (SEQ ID NO: 9) is in written text font and amino acid sequenceof Pritumumab light chain (SEQ ID NO: 36) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 54 shows the amino acid sequence of Cantuzumab—HC-PD1 andLC-TGFβRII fusion protein wherein the amino acid sequence for theCantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for PD1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font and amino acid sequenceof Cantuzumab light chain (SEQ ID NO: 30) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 55 shows the amino acid sequence of Cixutumumab—HC-PD1 andLC-TGFβRII fusion protein wherein the amino acid sequence for theCixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for PD1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font and amino acid sequenceof Cixutumumab light chain (SEQ ID NO: 32) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 56 shows the amino acid sequence of Clivatuzumab—HC-PD1 andLC-TGFβRII fusion protein wherein the amino acid sequence for theClivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for PD1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font and amino acid sequenceof Clivatuzumab light chain (SEQ ID NO: 34) is attached to aminoresidues for TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4)identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

FIG. 57 shows the amino acid sequence of Pritumumab—HC-PD1 andLC-TGFβRII fusion protein wherein the amino acid sequence for thePritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ IDNO: 3) shown in italics and the sequence for PD1 (immunomodulatorymoiety) (SEQ ID NO: 10) is in written text font and amino acid sequenceof Pritumumab light chain (SEQ ID NO: 36) is attached to amino residuesfor TGF-βRII (immunomodulatory moiety) (SEQ ID NO: 4) identified in boldletters with a linker (SEQ ID NO: 3) therebetween.

FIG. 58 shows the amino acid sequence of Cantuzumab HC-TGFβRII-4-1BBfusion protein wherein the amino acid sequence for Cantuzumab heavychain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text fontwith linker between (SEQ ID No: 11) and including the amino acidsequence of Cantuzumab light chain (SEQ ID NO: 30).

FIG. 59 shows the amino acid sequence of Cixutumumab HC-TGFβRII-4-1BBfusion protein wherein the amino acid sequence for Cixutumumab heavychain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text fontwith linker between (SEQ ID No: 11) and including the amino acidsequence of Cixutumumab light chain (SEQ ID NO: 32).

FIG. 60 shows the amino acid sequence of Clivatuzumab HC-TGFβRII-4-1BBfusion protein wherein the amino acid sequence for Clivatuzumab heavychain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text fontwith linker between (SEQ ID No: 11) and including the amino acidsequence of Clivatuzumab light chain (SEQ ID NO: 34).

FIG. 61 shows the amino acid sequence of Pritumumab HC-TGFβRII-4-1BBfusion protein wherein the amino acid sequence for Pritumumab heavychain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text fontwith linker between (SEQ ID No: 11) and including the amino acidsequence of Pritumumab light chain (SEQ ID NO: 36).

FIG. 62 shows the amino acid sequence of Cantuzumab HC-TGFβRII-PD1fusion protein wherein the amino acid sequence for Cantuzumab heavychain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for PD1(immunomodulatory moiety) (SEQ ID NO: 10) is in written text font withlinker between (SEQ ID No: 11) and including the amino acid sequence ofCantuzumab light chain (SEQ ID NO: 30).

FIG. 63 shows the amino acid sequence of Cixutumumab HC-TGFβRII-PD1fusion protein wherein the amino acid sequence for Cixutumumab heavychain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for PD1(immunomodulatory moiety) (SEQ ID NO: 10) is in written text font withlinker between (SEQ ID No: 11) and including the amino acid sequence ofCixutumumab light chain (SEQ ID NO: 32).

FIG. 64 shows the amino acid sequence of Clivatuzumab HC-TGFβRII-PD1fusion protein wherein the amino acid sequence for Clivatuzumab heavychain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for PD1(immunomodulatory moiety) (SEQ ID NO: 10) is in written text font withlinker between (SEQ ID No: 11) and including the amino acid sequence ofClivatuzumab light chain (SEQ ID NO: 34).

FIG. 65 shows the amino acid sequence of Pritumumab HC-TGFβRII-PD1fusion protein wherein the amino acid sequence for Pritumumab heavychain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown initalics and the sequence for TGFβRII (immunomodulatory moiety) (SEQ IDNO: 4) is identified in bold letters and the amino acid sequence for PD1(immunomodulatory moiety) (SEQ ID NO: 10) is in written text font withlinker between (SEQ ID No: 11) and including the amino acid sequence ofPritumumab light chain (SEQ ID NO: 36).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See, e.g., Sambrook, et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS INENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane,eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. The terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. As used in the description of theinvention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. The following terms have themeanings given:

The term “polynucleotide” as used herein means a sequence of nucleotidesconnected by phosphodiester linkages. Polynucleotides are presentedherein in the direction from the 5′ to the 3′ direction. Apolynucleotide of the present invention can be a deoxyribonucleic acid(DNA) molecule or ribonucleic acid (RNA) molecule. Where apolynucleotide is a DNA molecule, that molecule can be a gene or a cDNAmolecule. Nucleotide bases are indicated herein by a single letter code:adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) anduracil (U). A polynucleotide of the present invention can be preparedusing standard techniques well known to one of skill in the art.

The term, “optimized” as used herein means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence. The optimizedsequences herein have been engineered to have codons that are preferredin CHO mammalian cells; however optimized expression of these sequencesin other eukaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized. The term “expression” as used herein is defined as thetranscription and/or translation of a particular nucleotide sequencedriven by its promoter.

The term “transfection” of a cell as used herein means that geneticmaterial is introduced into a cell for the purpose of geneticallymodifying the cell. Transfection can be accomplished by a variety ofmeans known in the art, such as transduction or electroporation.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, ocular cancer, pancreatic cancer, colorectalcancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia,lung cancer and the like.

The term “transgene” is used in a broad sense to mean any heterologousnucleotide sequence incorporated in a vector for expression in a targetcell and associated expression control sequences, such as promoters. Itis appreciated by those of skill in the art that expression controlsequences will be selected based on ability to promote expression of thetransgene in the target cell. An example of a transgene is a nucleicacid encoding a chimeric fusion protein of the present invention.

The term “expression vector” as used herein means a vector containing anucleic acid sequence coding for at least part of a gene product capableof being transcribed. Expression vectors can contain a variety ofcontrol sequences, which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operatively linkedcoding sequence in a particular host organism. In addition to controlsequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well. The term also includes a recombinant plasmid or virusthat comprises a polynucleotide to be delivered into a host cell, eitherin vitro or in vivo. Preferably the host cell is a transient cell lineor a stable cell line and more preferably a mammalian host cell andselected from the group consisting of HEK293, CHO and NSO.

The term “subject,” as used herein means a human or vertebrate animalincluding a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey,rat, and mouse.

The term “therapeutically effective amount” as used herein means theamount of the subject compound that will elicit the biological ormedical response of a tissue, system, animal or human that is beingsought by the researcher, veterinarian, medical doctor or otherclinician.

The term “pharmaceutically acceptable” as used herein means the carrier,diluent or excipient must be compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

The term “recombinant” as used herein means a genetic entity distinctfrom that generally found in nature. As applied to a polynucleotide orgene, this means that the polynucleotide is the product of variouscombinations of cloning, restriction and/or ligation steps, and otherprocedures that result in the production of a construct that is distinctfrom a polynucleotide found in nature.

The term “substantial identity” or “substantial similarity,” as usedherein when referring to a nucleic acid or fragment thereof, indicatesthat when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 95 to 99% of thesequence.

The term “peptide,” “polypeptide” and “protein” are used interchangeablyto denote a sequence polymer of at least two amino acids covalentlylinked by an amide bond.

The term “homologous” as used herein and relating to peptides refers toamino acid sequence similarity between two peptides. When an amino acidposition in both of the peptides is occupied by identical amino acids,they are homologous at that position. Thus by “substantially homologous”means an amino acid sequence that is largely, but not entirely,homologous, and which retains most or all of the activity as thesequence to which it is homologous. As used herein, “substantiallyhomologous” as used herein means that a sequence is at least 50%identical, and preferably at least 75% and more preferably 95% homologyto the reference peptide. Additional peptide sequence modification areincluded, such as minor variations, deletions, substitutions orderivitizations of the amino acid sequence of the sequences disclosedherein, so long as the peptide has substantially the same activity orfunction as the unmodified peptides. Notably, a modified peptide willretain activity or function associated with the unmodified peptide, themodified peptide will generally have an amino acid sequence“substantially homologous” with the amino acid sequence of theunmodified sequence.

The term “administering” as used herein is defined as the actualphysical introduction of the composition into or onto (as appropriate)the host subject. Any and all methods of introducing the compositioninto the subject are contemplated according to the present invention;the method is not dependent on any particular means of introduction andis not to be so construed. Means of introduction are well-known to thoseskilled in the art, and preferably, the composition is administeredsubcutaneously or intratumorally. One skilled in the art will recognizethat, although more than one route can be used for administration, aparticular route can provide a more immediate and more effectivereaction than another route. Local or systemic delivery can beaccomplished by administration comprising application or instillation ofthe immunovaccines into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, orintradermal administration. In the event that the tumor is in thecentral nervous system, the composition must be administeredintratumorally because there is no priming of the immune system in thecentral nervous system.

Although chemotherapeutic agents can induce “immunogenic” tumor celldeath and facilitate cross-presentation of antigens by dendritic cells,tumors create a tolerogenic environment that allows them to suppress theactivation of innate and adaptive immune responses and evade immunologicattack by immune effector cells. The present invention providesstrategies to counteract tumor-induced immune tolerance in the tumormicroenvironment and can enhance the antitumor efficacy of chemotherapyby activating and leveraging T cell-mediated adaptive antitumor immunityagainst disseminated cancer cells.

The present invention is based on the discovery that targetedimmunomodulatory antibodies or fusion proteins of the present inventioncan counteract or reverse immune tolerance of cancer cells. Cancer cellsare able to escape elimination by chemotherapeutic agents ortumor-targeted antibodies via specific immunosuppressive mechanisms inthe tumor microenvironment and such ability of cancer cells isrecognized as immune tolerance. By counteracting tumor-induced immunetolerance, the present invention provides effective compositions andmethods for cancer treatment, optional in combination with anotherexisting cancer treatment.

The present invention provides compositions and methods for producingfusion proteins that counteract immune tolerance in the tumormicroenvironment and promote T cell-mediated adaptive antitumor immunityfor maintenance of durable long-term protection against recurrent ordisseminated cancers. These fusion proteins are designed to facilitateeffective long term T cell-mediated immune responses against tumor cellsby at least one of the following:

a. promoting death of tumor cells via enhancement of antibody-dependentcellular cytotoxicity (ADCC); andb. increasing activation and proliferation of antitumor CD8+ T cells bynegating immune suppression mediated by regulatory T cells and myeloidsuppressor cells. These antitumor immune responses may be activated intandem with the sensitization of tumor cells to immune effector-mediatedcytotoxicity, thereby establishing a positive feedback loop thataugments tumor cytoreduction and reinforces adaptive antitumor immunity.

In addition, the fusion proteins of the present invention aredistinguished from and superior to existing therapeutic, molecules in atleast one of the following aspects: (i) To counteract immune tolerancein the tumor microenvironment and promote T cell-mediated adaptiveantitumor immunity for maintenance of long-term protection againstrecurrent or disseminated cancers (for prevention or treatment ofdiverse cancers); (ii) To produce immune cell compositions for adoptivecellular therapy of diverse cancers; and (iii) To serve as immuneadjuvants or vaccines for prophylaxis of diverse cancers or infectiousdiseases.

The targeted immunostimulatory antibodies and/or fusion proteins of theinvention provide the ability to disrupt immunosuppressive networks inthe tumor microenvironment. Tumors employ a wide array of regulatorymechanisms to avoid or suppress the immune response. Cancer cellsactively promote immune tolerance in the tumor microenvironment via theexpression of cytokines and molecules that inhibit the differentiationand maturation of antigen-presenting dendritic cells (DC). Theimmunosuppressive cytokines and ligands produced by tumor cells includethe following: (i) Transforming growth factor-beta (TGF-β); (ii)Programmed death-1 ligand 1 (PD-L1; B7-H1); (iii) Vascular endothelialgrowth factor (VEGF); and (iv) Interleukin-10 (IL-10).

In addition to blocking dendritic cell (DC) maturation, these moleculespromote the development of specialized subsets of immunosuppressive CD4⁺T cells (regulatory T cells; Treg cells) and myeloid-derived suppressorcells (MDSC). Tregs are a minority sub-population of CD4⁺ T cells thatconstitutively express CD25 [the interleukin-2 (IL-2) receptor cc-chain]and the forkhead box P3 (FOXP3) transcription factor. Tregs(CD4+CD25+FoxP3+ cells) maintain immune tolerance by restraining theactivation, proliferation, and effector functions of a wide range ofimmune cells, including CD4 and CDS T cells, natural killer (NK) and NKTcells, B cells and antigen presenting cells (APCs) in vitro and in vivo.

The accumulation of Treg cells in the tumor microenvironment reinforcestumor immune tolerance and facilitates tumor progression and metastases.The increased expression of immunosuppressive cytokines (TGF-β; PD-L1)and tumor-infiltrating Tregs is correlated with a reduction of survivalof patients with diverse types of cancers. The fusion proteins of thepresent invention inhibit key immunosuppressive molecules expressed bythe targeted tumor cell or tumor-infiltrating Treg cells and myeloidsuppressor cells (DCs or MDSC). As such, they provide the targetedability to inhibit the development or function of Tregs within the tumormicroenvironment.

The present invention provides a method of preventing or treating aneoplastic disease. The method includes administration to a subject inneed thereof one or more fusion proteins of the present invention incombination with another anticancer therapy, wherein the anticancertherapy is a chemotherapeutic molecule, antibody, small molecule kinaseinhibitor, hormonal agent, cytotoxic agent, targeted therapeutic agent,anti-angiogenic agent, ionizing radiation, ultraviolet radiation,cryoablation, thermal ablation, or radiofrequency ablation.

As used herein, the term “antibody” includes natural or artificial mono-or polyvalent antibodies including, but not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments. F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. Theantibody may be from any animal origin including birds and mammals. Inone aspect, the antibody is, or derived from, a human, murine (e.g.,mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse,or chicken. Further, such antibody may be a humanized version of anantibody. The antibody may be monospecific, bispecific, trispecific, orof greater multispecificity. The antibody herein specifically include a“chimeric” antibody in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

Examples of antibodies which can be incorporated into compositions andmethods disclosed herein include, but are not limited, to antibodiessuch as trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2mAb); cetuximab (chimeric monoclonal antibody to epidermal growth factorreceptor EGFR): panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFRantibody); Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb);MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanizedanti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptorbispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb);Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb);Tositumumab-1131 (anti-CD20 mAb); ibritumomab tiuxetan (anti-CD20 mAb);Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab(anti-VEGF mAb); Aflibercept (extracellular domains of YEGFR1 and VEGFR2fused to IgG1 Fc): AMG386 (angiopoietin-1 and -2 binding peptide fusedto IgG1 Fc); Dalotuzumab (anti-1GF-1R mAb): Gemtuzumab ozogamicin(anti-CD33 mAb); Alemtuzumab (anti-Campath-1/CD52 mAb); Brentuximabvedotin (anti-CD30 mAb); Catumaxomab (bispecific mAb that targetsepithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb);Girentuximab (anti-Carbonic anhydrase ix): or Farletuzumab (anti-folatereceptor). Other examples include antibodies such as Panorex™ (17-1 A)(murine monoclonal antibody); Panorex (@ (17-1 A) (chimeric murinemonoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope)(with BCG): Oncolym (Lym-1 monoclonal antibody); SMART M 1 95 Ab,humanized 13′ 1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotypic mousemAb); 3622W94 mAb that binds to EGP40 (17-1 A) pancarcinoma antigen onadenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART M1 95Ab, humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab): TNT(chimeric mAb to histone antigens); TNT (chimeric mAb to histoneantigens); GJiomab-H (Monoclonals—Humanized Abs); GN1-250 Mab; EMD-72000(chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); andMDX-260 bispecific, targets GD-2, ANA Ab, SMART lDiO Ab, SMART ABL 364Ab or ImmuRAIT-CEA.

Various methods have been employed to produce antibodies. Hybridomatechnology, which refers to a cloned cell line that produces a singletype of antibody, uses the cells of various species, including mice(murine), hamsters, rats, and humans. Another method to prepare anantibody uses genetic engineering including recombinant DNA techniques.For example, antibodies made from these techniques include, amongothers, chimeric antibodies and humanized antibodies. A chimericantibody combines DNA encoding regions from more than one type ofspecies. For example, a chimeric antibody may derive the variable regionfrom a mouse and the constant region from a human. A humanized antibodycomes predominantly from a human, even though it contains nonhumanportions. Like a chimeric antibody, a humanized antibody may contain acompletely human constant region. But unlike a chimeric antibody, thevariable region may be partially derived from a human. The nonhuman,synthetic portions of a humanized antibody often come from CDRs inmurine antibodies. In any event, these regions are crucial to allow theantibody to recognize and bind to a specific antigen.

In one embodiment, a hybridoma can produce a targeted fusion proteincomprising a targeting moiety and an immunomodulatory moiety. In oneembodiment, a targeting moiety comprising an antibody, antibodyfragment, or polypeptide is linked or fused to an immunomodulatorymoiety consisting of a polypeptide, with a linker or without a linker.The linker can be an amino acid linker. In one embodiment, a linker is(GGGGS)n wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. For example,GGGGSGGGGSGGGGS (SEQ ID NO: 3). In another embodiment, a linker isEPKSCDK (SEQ ID NO: 11). In various aspects, the length of the linkermay be modified to optimize binding of the target moiety or the functionof the immunomodulatory moiety. In various aspects, the immunomodulatorymoiety is a polypeptide that is fused to the C-terminus of the Fc regionof the heavy chain of a targeting antibody or Fc-containing fusionprotein. In another aspect, the immunomodulatory moiety is a polypeptidethat is fused to the C-terminus of the light chain of a targetingantibody.

An antibody fragment can include a portion of an intact, antibody, e.g.including the antigen-binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; Fcfragments or Fc-fusion products; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragment(s). An intact antibody is one which includes anantigen-binding variable region as well as a light chain constant domain(CL) and heavy chain constant domains, CHI, CH2 and CH3. The constantdomains may be native sequence constant domains (e.g., human nativesequence constant domains) or amino acid sequence variant thereof forany other modified Fc (e.g. glycosylation or other engineered Fc).

The fusion proteins of the present invention may be synthesized byconventional techniques known in the art, for example, by chemicalsynthesis such as solid phase peptide synthesis. Such methods are knownto those skilled in the art. In general, these methods employ eithersolid or solution phase synthesis methods, well known in the art.Specifically, the methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups and any solid supportare removed either sequentially or concurrently to afford the finalpolypeptide. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under condition that do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide.

Typical protecting groups include t-butyloxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc), benxyloxycarbonyl (Cbz),p-toluenesulfonyl (Tos); 2,4-dinitrophenyl, benzyl (Bzl),biphenylisopropyloxy-carboxycarbonyl, cyclohexyl, isopropyl, acetyl,o-nitrophenylsulfonyl, and the like. Of these, Boc and Fmoc arepreferred.

Typical solid supports are generally cross-linked polymeric materials.These include divinylbenzene cross-linked styrene-based polymers, forexample, divinylbenzene-hydroxymethyl styrene copolymers,divinylbenzene-chloromethyl styrene copolymers, anddivinylbenzene-benzhydrylaminopolystyrene copolymers. Thedivinylbenzene-benzhydrylaminopolystyrene copolymers, as illustratedherein using p-methyl-benzhydrylamine resin, offers the advantage ofdirectly introducing a terminal amide functional group into the peptidechain, which function is retained by the chain when the chain is cleavedfrom the support.

In one method, the polypeptides are prepared by conventional solid phasechemical synthesis on, for example, an Applied Biosystems, Inc. (ABI)430A peptide synthesizer using a resin that permits the synthesis of theamide peptide form and using t-Boc amino acid derivatives (PeninsulaLaboratories, Inc.) with standard solvents and reagents. Polypeptidescontaining either L- or D-amino acids may be synthesized in this manner.Polypeptide composition is confirmed by quantitative amino acid analysisand the specific sequence of each peptide may be determined by sequenceanalysis.

Preferably, the polypeptides can be produced by recombinant DNAtechniques by synthesizing DNA encoding the desired polypeptide. Oncecoding sequences for the desired polypeptides have been synthesized orisolated, they can be cloned into any suitable vector for expression.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice. Thegene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. Heterologous leader sequences can be addedto the coding sequence that causes the secretion of the expressedpolypeptide from the host organism. Other regulatory sequences may alsobe desirable which allow for regulation of expression of the proteinsequences relative to the growth of the host cell. Such regulatorysequences are known to those of skill in the art, and examples includethose which cause the expression of a gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence of aregulatory compound. Other types of regulatory elements may also bepresent in the vector, for example, enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

The expression vector may then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkeykidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, NOS cells derived fromcarcinoma cells, such as sarcoma, as well as others. Similarly,bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcusspp., will find use with the present expression constructs. Yeast hostsuseful in the present invention include inter alia, Saccharomycescerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha,Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii,Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni. The proteinsmay also be expressed in Trypanosomes.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto growth media, the protein can be purified directly from the media.If the protein is not secreted, it is isolated from cell lysates. Theselection of the appropriate growth conditions and recovery methods arewithin the skill of the art. Once purified, the amino acid sequences ofthe proteins can be determined, i.e., by repetitive cycles of Edmandegradation, followed by amino acid analysis by HPLC. Other methods ofamino acid sequencing are also known in the art.

Once synthesized or otherwise produced, the inhibitory activity of acandidate polypeptide can be tested by assessing the ability of thecandidate to inhibit the lipopolysaccharide-induced nucleartranslocation of NF-.kappa.B by, for example, using murine endothelialcells.

The fusion proteins of the present invention can be formulated intotherapeutic compositions in a variety of dosage forms such as, but notlimited to, liquid solutions or suspensions, tablets, pills, powders,suppositories, polymeric microcapsules or microvesicles, liposomes, andinjectable or infusible solutions. The preferred form depends upon themode of administration and the particular cancer type targeted. Thecompositions also preferably include pharmaceutically acceptablevehicles, carriers or adjuvants, well known in the art, such as humanserum albumin, ion exchangers, alumina, lecithin, buffer substances suchas phosphates, glycine, sorbic acid, potassium sorbate, and salts orelectrolytes such as protamine sulfate. Suitable vehicles are, forexample, water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. Actual methods of preparing such compositions areknown, or will be apparent, to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 18th edition, 1990.

The above compositions can be administered using conventional modes ofdelivery including, but not limited to, intravenous, intraperitoneal,oral, intralymphatic, or subcutaneous administration. Localadministration to a tumor in question, or to a site of inflammation,e.g., direct injection into an arthritic joint, will also find use withthe present invention.

Therapeutically effective doses will be easily determined by one ofskill in the art and will depend on the severity and course of thedisease, the patient's health and response to treatment, and thejudgment of the treating physician.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1

The Fusion proteins comprising of IgG heavy chain linked toimmunomodulator (either suppressor or activator) ligands were expressedby codon optimized genes for the expression of CHO cells. The codonoptimized nucleotide sequences defined by SEQ ID NOs: 12 to 28 wereexpressed in (CHO) cells and the expressed chimeric/fusion proteins areshown in Table 1

Fusion protein Details Anti-HER2/neu heavy chain + TGFβ-RII ECD andAnti-HER2/neu light chain Anti-EGFR1 heavy chain + TGFβ-RII ECD andAnti- EGFR1 light chain Anti-CTLA4 heavy chain + TGFβ-RII ECD andAnti-CTLA4 light chain Anti-CTLA4 heavy chain + PD1 ectodomain andAnti-CTLA4 light chain Anti-HER2/neu heavy chain + 4-1BBL andAnti-HER2/neu light chain Anti-EGFR1 heavy chain + 4-1BBL and Anti-EGFR1 light chain Anti-CTLA4 heavy chain + 4-1BBL and Anti-CTLA4 lightchain PD1 ectodomain-Fc-4-1BBL TGFβRII ECD-Fc-4-1BBL Anti-EGFR1 heavychain + PD1 ectodomain and Anti- EGFR1 light chain Anti-CD20 heavychain + 4-1BBL and Anti- CD20 light chain Anti-HER2/neu heavy chain +PD1 ectodomain and Anti-HER2/neu light chain Anti-IL6Rheavy chain + PD1ectodomain and Anti-IL6R light chain Anti-IL6Rheavy chain + TGFβ-RII ECDand Anti-IL6R light chain Anti-4-1BB heavy chain + PD1 ectodomain andAnti-4-1BB light chain

The expressed protein were characterized by using SDS PAGE and theexpressed fusion proteins Anti-HER2/neu-TGFβRII and Anti-EGFR1-TGFβRIIwere purified from culture supernatants using ProteinA column and theresults are shown in FIG. 22. Notably, Anti-EGFR1-TGFβRII light chainmass is higher and it may be because of the presence of twoglycosylation sites on the variable regions light and heavy chain. Boththe Anti-HER2/neu-TGFβRII & Anti-EGFR1-TGFβRII heavy chains mass arehigher because of the TGFβRII. Also Anti-HER2/neu-TGFβRII heavy chainhas four N-glycosylation sites while Anti-EGFR1-TGFβRII has fiveN-glycosylation sites.

Example 2

Protein A/SEC chromatography. The Anti-HER2/neu-TGFβRII andAnti-EGFR1-TGFβRII samples were analyzed by ProteinA/SEC chromatographyand the results are shown in FIG. 23. FIG. 23 A shows a sharp peak ofelution of Bmab200(Herceptin) vs a broader elution peak is believed tobe a measure of heterogeneity due to presence of glycosylation as thereare three additional N-glycosylation sites that are present in theTGFβRII region. Notably storage at −80 C did not causing aggregation.The shift in the position or appearance of the peak early in SEC columnindicates that the increase in the molecular weight is because of thefusion partner. This once again confirms that the full length moleculeis being expressed. FIG. 23 B shows a sharp peak of elution ofBmab200(Herceptin) vs a broader elution peak which is believed to be ameasure of heterogeneity due to presence of glycosylation sites as thereare three additional N-glycosylation sites are present in the TGFβRIIregion. Again, storage at −80 C did not causing aggregation. The shiftin the position or appearance of the peak early in SEC column indicatesthat the increase in the molecular weight is because of the fusionpartner. This once again confirms that the full length molecule is beingexpressed.

Example 3

Functional assays for the Fusion proteins. ELISA experiment was carriedout to check the binding ability of Anti-HER2/neu-TGFβRII andAnti-EGFR1-TGFβRII to TGFβ. FIG. 24 A shows that Anti-HER2/neu-TGFβRIIand Anti-EGFR1-TGFβRII molecules bind to the TGFβ indicating that thefusion protein is functional. FIG. 24 B shows that Anti-HER2-TGFβRIIinhibits the proliferation of BT474 cell line similar to the Bmab200(Herceptin). FIG. 25 shows that Anti-EGFR1-TGFβRII-inhibits theproliferation of A431 cell line similar to the Cetuximab.

Example 4

Antibody dependent cellular cytotoxicity ADCC activity forAnti-HER2/neu-TGFβRII fusion protein was conducted to determine that theprotein binds to the target receptors on the cells. The results areshown in FIG. 26 wherein the activity is determined in BT474 cells andit is evident that ADCC activity (% lysis of cells) of Anti-HER2-TGFβRIIon BT474 cells is similar to that of Bmab200(Herceptin). FIG. 27 showsADCC activity of Anti-EGFR1-TGFβRII on A431 cells wherein the ADCCactivities are similar to that of Cetuximab. FIG. 28 shows the ADCCactivity of ADCC activity of Anti-EGFR1-4-1BB in comparison withAnti-EGFR1-TGFβRII and cetuximab.

Example 5

Binding Activity of the expressed proteins. The aim of this assay is totest the functionality of the fusion proteins to bind to the targetreceptors on the cells in a dose dependent manner. FIG. 29 A shows thatthe binding activity of Anti-CTLA4-TGFβRII to TGFβ1 is comparable toAnti-EGFR1-TGFβRII and B shows that the binding activity ofAnti-CTLA4-TGFβRII to CTLA4. FIG. 30 A shows the binding activity ofAnti-CTLA4-TGFβRII to determine the level of PD1-Fc binding and B showsthe binding activity of Anti-EGRF1-4-1BB to determine the binding of4-1BBL. FIG. 31 A shows the binding activity of Anti-EGFR1-4-1BB to EGFRand B shows the binding activity of PD1-Fc-4-1BB to find out PDL1-Fc.FIG. 32 shows the binding activity of Anti-EGFR1-PD1 to EGFR and PD1.

Example 6

Confirmation of primary structure of molecule. As shown in FIG. 33, theexpressed proteins are evaluated to determine the molecular weight andthe presence of glycosylation. The samples were analyzed by reducing andnon-reducing SDS PAGE. The heavy and light chains of the antibody areseparated by reduction alkylation so that the reduced structures can beevaluated. Tryptic digestion of the fusion proteins provides for theidentification of the primary sequence. MS/MS analysis of the proteinsis performed.

Mass Spectrometry Analysis of Anti-HER2/neu-TGFβRII andAnti-EGFR1-TGFβRII. The fusion protein shown in FIG. 1 was expressed andtested. FIG. 34 A shows the mass spectrum Mass Spectrum of light chain(LC)(Reduced) of Anti-HER2/neu-TGFβRII ECD fusion and B showsDeconvoluted Mass Spectrum of LC (Reduced) of Anti-HER2/neu-TGFβRII ECDfusion. FIG. 35 shows the Mass Spectrum of heavy chain (HC) (Reduced) ofAnti-HER2/neu-TGFβRII ECD fusion.

The fusion protein shown in FIG. 2 was expressed and tested. FIG. 36 Ashows the Mass Spectrum of LC (Reduced) of Anti-EGFR1-TGFβRII ECD and Bshows the Deconvoluted Mass Spectrum of LC (Reduced) ofAnti-EGFR1-TGFβRII ECD. FIG. 37 shows the Mass Spectrum of HC (Reduced)of Anti-EGFR1-TGFβRII ECD.

Example 7

The fusion proteins having amino acid sequences as described in FIGS. 1and 2 were inspected using UV chromatography and providing chromatogramsresulting from the chromatographic separation of the tryptic digest ofthe fusion proteins and tested with UV 218-222 nm wavelength. Total IonCurrent (TIC) corresponding to UV trace was also evaluated. FIG. 38 Ashows the UV Chromatogram of Tryptic Peptides of Anti-HER2/neu-TGFβRIIECD fusion protein and B shows the Total Ion Chromatogram (TIC) ofTryptic Peptides of Anti-HER2/neu-TGFβRII ECD fusion protein. FIGS. 39,40 and 41 provide lists of expected/observed tryptic peptide of thelight chain, heavy chain and linked motif of the Anti-HER2/neu-TGFβRIIECD fusion protein, respectively. Notably, all the expected peptides ofthe molecules were identified including the light and heavy chainpeptides and the peptides of the linked motif (TGF βRII).

FIG. 42 A shows the UV Chromatogram of Tryptic Peptides ofAnti-EGFR1-TGFβRII ECD fusion protein and B shows the Total IonChromatogram (TIC) of Tryptic Peptides of Anti-EGFR1-TGFβRII ECD fusionprotein. FIGS. 43, 44, and 45 provide lists of expected/observed trypticpeptide of the light chain, heavy chain and linked motif of theAnti-EGFR1-TGFβRII ECD fusion protein, respectively. Again all theexpected peptides of the molecules were identified including the lightand heavy chain peptides and the peptides of the linked motif (TGFβRII).

Example 8

The host cell line used for the expression of recombinant fusion proteinexpression is CHO cells or the derivative of the CHO cells. The CHOcells referred here is either freedom CHO-S cells; CHO-S Cells areCHO-derived cells adapted to high density, serum-free suspension culturein chemically-defined medium that are capable of producing high levelsof secreted, recombinant protein or CHO K1 cells; having the same asATCC No. CCL-61. It is basically an adherent cell line. The vectors usedfor stable cell line:

The Freedom pCHO 1.0 vector, designed by ProBioGen AG, to express one ortwo genes of interest downstream of the vector's two different hybridCMV promoters. This vector contains the dihydrofolate reductase (DHFR)selection marker and a puromycin resistance gene, allowing selectionusing MTX and Puromycin simultaneously.

The light chain or the light chain fusion protein coding nucleic acidsequences are cloned into the restriction enzyme sites AvrII and BstZ17under the control of EF2/CMV promoter. The heavy chain or the heavychain fusion protein coding nucleic acid sequences are cloned, inrestriction enzyme sites EcoRV and Pad under the control of CMV/EF1promoter.

The construct(s) are transfected into Freedom CHO-S cells/CHOK1 cells.The high producer single, clonal cell strain is selected for producingthe recombinant fusion protein. Prepare the MCB and characterize forcell viability, productivity, stability and other parameters. The cellsare used for culturing followed by purification.

Example 9

The cell culture is performed in feed-batch mode. In the cell culture,the mammalian host cells used is Chinese Hamster Ovary (CHO) cells andculture medium are supplied initially. The CHO cells are geneticallyengineered to produce the Antibody-peptide fusion protein. The zincsulphate hepta hydrate salt is added in the medium at a concentration of0.4 mM. In contrast, there is no addition of any zinc salt in thecontrol medium. The production fermentation run starts with an initialcell count of 0.3-0.45×10⁶ cells/ml at 37±1° C., the first 3-4 days arededicated to grow the cells in batch phase. Next step involves loweringthe temperature to 31±1° C. and continuing the run till 7th day. Lactatereduces by almost 10-40% throughout the run. The produced fusion proteinis then collected from the media using the technique of affinitychromatography.

Example 10

The cell culture is performed in a feed-batch mode is employed. In thecell cultures the mammalian host cells and culture medium which isHyclone CDM4Mab are supplied initially. The salts (zinc) is also addedin the medium (0.3 mM). The production fermentation run starts with aninitial cell count of 0.3-0.45×10⁶ cells/nil at 37±1° C., the first 3-4days are dedicated to growing the cells in batch phase. Next stepinvolves lowering the temperature to 31+1−1° C. and continuing the runtill 7th day.

Example 11

Purification of antibody-peptide fusion immunostimulatory moleculesusing protein A column. Supernatant culture secreted from recombinantCHO cell line containing the fusion monoclonal antibodies is tested fortiter and endotoxins under sterile conditions. The supernatant issubjected to affinity chromatography using Mab Select Xtra Protein Aaffinity resin, washed and equilibrated with binding buffer. The pH ofthe supernatant is adjusted using 0.5M phosphate to the same pH as thecolumn; the supernatant is allowed to bind to the column/pass throughthe column at the flow rate of 0.5 ml/minute to achieve the maximumbinding. All the Antibody-proteins fusion molecules bind through the Fcregion while impurities are eliminated as flow through. The column iswashed with equilibration buffer and the bound fusion molecules areeluted using 0.1 M glycine at pH 3.0. The pH of the eluted proteins isadjusted to neutral pH or the stable formulation pH and the purifiedprotein are stored at −20° C. or at 2-8° C.

Example 12

Differentiating Trastuzumab from Trastuzumab-TGF βRII Receptor FusionMolecule

A breast cancer tumor overexpressing the ErbB2 receptor will either byconstitutive activation or heterodimerization with other members of theErbB family of receptors lead to tumor progression. This will involvethe binding of growth factors associated with the ErbB signalingpathway. In addition to this, the tumor creates a milieu wherein theimmune system is suppressed by activating TGF β and specific cytokinesinvolved in the subdued immune response. A novel molecule is generatedwherein Trastuzumab (anti ErbB2) is fused with the TGF βRII receptor asa fusion protein. While it is hypothesized that Trastuzumab will act asa targeted molecule homing into the ErbB2 overexpressing breast cancercells, the TGFβRII receptor will sequester TGFβ leading to immuneactivation. The experiment will utilize the growth of Herceptinresistant ErbB2 expressing cell lines (selected by growing BT474 cellsin the presence of Herceptin) in the presence of TGFβ, cytotoxic CD8positive cells and NK cells. While Trastuzumab will be ineffective ininducing cytotoxicity Trastuzumab TGFβRII receptor fusion molecule willsequester the TGFβ thereby preventing the inhibition of cytotoxic CD8and NK cells. This will lead to enhanced cytotoxicity observed inTrastuzumab—TGFβRII receptor fusion treated cells over cells treatedwith Trastuzumab alone. The readout for the experiment will use AlamarBlue a resazurin dye which will get activated directly proportional tolive cells present. Another method could be to measure cytotoxicity byusing cytotox glo which measures protease release which directlycorresponds to proportional dead cells. Yet another method could be theuse of the flow cytometer directly measuring apoptotic and necrotic cellpopulation by using Annexin V and propidium iodide. Results from thesemultiple experiments will elucidate understanding of the activity of theconjugate molecule as compared to Trastuzumab alone.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

That which is claimed is:
 1. A chimeric fusion protein having bispecificbinding comprising at least one targeting moiety to target a cancer celland at least one immunomodulating moiety that counteracts immunetolerance, wherein the targeting moiety and the immunomodulating moietyare linked by an amino acid spacer of sufficient length of amino acidresidues so that both moieties can successfully bind to their individualtargets, wherein the immunomodulating moiety is PD-1 consisting of aminoacid sequence of SEQ ID NO: 10; wherein the amino acid spacer isselected from SEQ ID NO: 3 and SEQ ID NO: 11; and wherein the targetingmoiety is an anti-CTLA-4 antibody consisting of a heavy chain of SEQ IDNO: 7 and a light chain of SEQ ID NO: 8, wherein SEQ ID NO: 4 isattached via the amino acid spacer to the C-terminus of SEQ ID NO: 7 orSEQ ID NO: 8 of the Anti-CTLA-4 antibody.
 2. A method of increasinglysis of cancer cells, the method comprising contacting cancer cellswith a therapeutic amount of a chimeric fusion protein, wherein thechimeric fusion protein comprises a targeting moiety to target a cancercell and an immunomodulating moiety that counteracts immune tolerance,wherein the targeting moiety and the immunomodulating moiety are linkedby an amino acid spacer of sufficient length of amino acid residues sothat both moieties can successfully bind to their individual targets,wherein the immunomodulating moiety is SEQ ID NO: 10, wherein the aminoacid spacer is selected from SEQ ID NO: 3 or SEQ ID NO: 11; and whereinthe targeting moiety Anti-CTLA4 antibody is heavy chain SEQ ID NO: 7 andlight chain SEQ ID NO: 8; wherein SEQ ID NO: 10 is attached via theamino acid spacer to the C-terminus of SEQ ID NO 7 or SEQ ID NO:
 8. 3.The method of claim 2, wherein the cancer cells are in a subject in needof treatment.
 4. The method of claim 2, further comprising administeringthe chimeric fusion protein in combination with another existing cancertreatment.
 5. The method of claim 2, wherein suppression of an immunesystem of the subject in the cancer cell is reduced.
 6. The method ofclaim 2, wherein lysis of cancer cells is increased.
 7. The method ofclaim 2, wherein SEQ ID NO: 10 binds to PD-L1.